Hepmarc: A 96 week randomised controlled feasibility trial of add-on maraviroc in people with HIV and non-alcoholic fatty liver disease

Objectives Maraviroc may reduce hepatic inflammation in people with HIV and non-alcoholic fatty liver disease (HIV-NAFLD) through CCR5-receptor antagonism, which warrants further exploration. Methods We performed an open-label 96-week randomised-controlled feasibility trial of maraviroc plus optimised background therapy (OBT) versus OBT alone, in a 1:1 ratio, for people with virologically-suppressed HIV-1 and NAFLD without cirrhosis. Dosing followed recommendations for HIV therapy in the Summary of Product Characteristics for maraviroc. The primary outcomes were safety, recruitment and retention rates, adherence and data completeness. Secondary outcomes included the change in Fibroscan-assessed liver stiffness measurements (LSM), controlled attenuation parameter (CAP) and Enhanced Liver Fibrosis (ELF) scores. Results Fifty-three participants (53/60, 88% of target) were recruited; 23 received maraviroc plus OBT; 89% were male; 19% had type 2 diabetes mellitus. The median baseline LSM, CAP & ELF scores were 6.2 (IQR 4.6–7.8) kPa, 325 (IQR 279–351) dB/m and 9.1 (IQR 8.6–9.6) respectively. Primary outcomes: all individuals eligible after screening were randomised; there was 92% (SD 6.6%) adherence to maraviroc [target >90%]; 83% (95%CI 70%-92%) participant retention [target >65%]; 5.5% of data were missing [target <20%]. There were noo Serious Adverse Reactions; mild-moderate intensity Adverse Reactions were reported by five participants (5/23, 22% (95%CI 5%-49%)) [target <10%]. All Adverse Reactions resolved. Secondary outcomes: no important differences were seen by treatment group for the change from baseline in LSM, CAP or ELF scores Conclusions This feasibility study provides preliminary evidence of maraviroc safety amongst people with HIV-NAFLD, and acceptable recruitment, retention, and adherence rates. These data support a definitive randomised-controlled trial assessing maraviroc impact on hepatic steatosis and fibrosis. Trial registration Clinical trial registry: ISCRTN, registration number 31461655.

Introduction Non-alcoholic fatty liver disease (NAFLD), defined as fat accumulation in �5% of hepatocytes without a secondary cause, [1] may progress to steatohepatitis (NASH), fibrosis, cirrhosis and hepatocellular carcinoma. NAFLD represents one of the commonest causes of liver disease in people with HIV (PWH), with a prevalence of 35% in a systematic review, but varying from 13% to 73% [2][3][4]. Progression to NASH and hepatic fibrosis may be more likely in PWH with NAFLD (HIV-NAFLD) than in HIV-negative individuals with NAFLD (primary NAFLD) [5]. In a meta-analysis, metabolic abnormalities were risk factors for HIV-NAFLD, including type 2 diabetes mellitus, hypertension, and elevated body mass index (BMI) [2]. HIV-related factors, such as metabolic effects of viral proteins, immune activation, gut microbial translocation, and antiretrovirals, may also play a role, although their contribution is not well understood [4,6].
Lifestyle changes, including physical activity and dietary modification, are the mainstay of therapy [7] for HIV-NAFLD, but are difficult to achieve [8]. Pharmacological therapies are urgently required but few trials have investigated potential candidates, especially in PWH [1]. In a randomised controlled trial (RCT), the growth hormone-releasing hormone analogue, tesamorelin, reduced hepatic fat fraction in HIV-NAFLD; however, it is given by subcutaneous injection, limiting its use [9]. In a small, single-arm trial, oral vitamin E showed efficacy in HIV-NAFLD, although there are concerns over long-term safety [1,10].
Chemokine (C-C motif) ligand 5 (CCL5)/ Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES), the ligand for C-C chemokine receptor type 5 (CCR5), plays a key role in hepatic inflammation. CCR5 mediates intrahepatic immune cell interactions which promote activation and migration of Kupffer cells and hepatic stellate cells; these in turn promote inflammation and fibrosis [11,12]. Antagonism of this pathway could therefore reduce fibrosis progression [13,14].
The CCR5 receptor antagonist, maraviroc (MVC), is licensed for HIV-1 treatment as part of combination antiretroviral therapy (cART) in therapy-naïve and -experienced individuals, where the infecting strain is CCR5 tropic [15,16]. MVC inhibits HIV-1 gp120 binding to the CCR5 co-receptor, preventing virus entry into the cell. Its antagonism of CCL5-CCR5-mediated interactions raises the possibility of additional anti-inflammatory effects. Data from in vitro studies, mouse models, and retrospective HIV/HCV and HIV-NAFLD cohorts, suggest a reduction in hepatic fibrosis progression [17][18][19][20]. In an RCT, cenicriviroc, a CCR2 and CCR5 antagonist, reduced hepatic fibrosis in primary NAFLD [21].
MVC therefore represents a potential treatment for HIV-NAFLD. However, dosing is usually twice daily, unlike currently recommended antiretrovirals. We therefore aimed to conduct a RCT to evaluate the safety, acceptability and feasibility of MVC add-on therapy to cART in HIV-NAFLD in preparation for conducting a larger RCT in the future. The RCT was successfully completed and findings are presented below.

Study design and participants
This was a phase IV, open-label, dual, parallel arm, randomised, multi-centre, feasibility trial comparing MVC plus optimised background therapy (MVC+OBT) versus OBT alone in HIV-NAFLD over 96 weeks. The sites were hospitals in London (Barts, Chelsea and Westminster), the Midlands (Nottingham), North (Liverpool, South Tees), South (Brighton) and West (Bristol) of England.
Potentially eligible individuals were identified by members of the direct care team through review of clinic databases, pre-identification of those attending routine consultations and review of medical notes during follow-up. Anonymised information collected on individuals contacted but not randomised were age, gender, ethnicity, reason for lack of eligibility or if they were potentially eligible but declined.
Detailed methodology has been presented previously [22]. In brief, inclusion criteria included: provision of written, informed consent, �18 years of age, HIV-1 viral load (VL) <50 copies/ml for �6 months, evidence of NAFLD including hepatic steatosis on imaging (either ultrasound, CT or MRI of the liver) or liver biopsy, performed within 6 months of screening. Exclusion criteria included: severe cardiovascular disease (including known angina or history of myocardial infarction), postural hypotension, prior MVC exposure, other causes of liver disease including viral, alcohol-associated, haemochromatosis, Wilson's disease, alpha-1 antitrypsin deficiency, drug-induced, autoimmune; cirrhosis (defined by histology, imaging, or liver stiffness (LSM) >13kPa); ALT or AST >205 IU/L (five times the upper limit of normal [23]); severe renal insufficiency (defined as creatinine clearance <30mL/min); HIV-2; MVC allergy or intolerance; co-medications contraindicated with MVC; pregnancy; breastfeeding.

Randomisation
Participants were randomised 1:1 in accordance with a computer-generated randomization schedule using Sealed Envelope [24] to receive MVC+OBT versus OBT. Randomisation was stratified according to: (1) current or past history of �6 months' exposure to a protease inhibitor (PI) versus no current and <6 months prior exposure (2) BMI �25 versus <25 (3) diabetes mellitus status (4) current exposure to lipid-lowering agents. This was due to reported positive associations for (1-3) with hepatic fibrosis progression [2,25] in HIV-NAFLD and a negative association for (4) with fibrosis progression in primary NAFLD [26,27]. In a cohort of 300 patients with HIV monitored with transient elastography for NAFLD development, current PI use was independently predictive of significant liver fibrosis (adjusted OR 3.96, 95% CI 1.64-9.54) [25]. In a systematic review of studies enrolling patients with HIV-NAFLD, elevated BMI and fasting glucose were independently associated with increased risk of significant liver fibrosis (Mean Difference (MD) 1.38, 95% CI 0.04-2.71, p = 0.004 and MD 0.80, 95% CI 0.47-1.13, p<0.00001 respectively) [2]. In primary NAFLD, current lipid lowering agent use was associated with protection from steatosis, NASH and fibrosis stages F2-F4 in a dose dependent manner (adjusted p<0.05 for all) [26]. As this was a feasibility study, the study was unblinded, with a plan for blinding within a larger RCT, should feasibility be demonstrated.

Procedures
MVC was prescribed twice daily at the licensed dose for HIV-1 therapy, adjusted according to co-medications according to the Summary of Product Characteristics. Participants attended every 24 weeks for 96 weeks plus a week 4 safety visit for those receiving maraviroc. All participants received advice on lifestyle and dietary modifications as standard of care interventions for HIV-NAFLD. Supplementary Table S1 in S1 File details the schedule of events.
In brief, the screening visit included history-taking, physical examination, height and weight measurement, laboratory investigations for eligibility, ECG, LSM and Controlled Attenuation Parameter (CAP) score measured by Fibroscan, and hepatic ultrasound scan. Fibroscan readings, HbA1c, CD4 T-lymphocyte count, and VL from screening were used as the baseline measures. This visit was performed by the site investigator or sub-investigator, who also confirmed eligibility. The baseline assessment included diet and exercise history, fasting bloods for glucose and lipids, Enhanced Liver Fibrosis (ELF) score and proviral DNA coreceptor tropism. There was an optional CT scan liver and spleen, and this was only offered at one site. Participants additionally completed questionnaires to evaluate quality of life (QoL): chronic liver disease questionnaire for NAFLD (CLDQ-NAFLD) [28], 36-Item Short Form Survey (SF-36) [29] and Work Productivity and Activity Impairment: Specific Health Problem (WPAI:SHP) [30].
Assessments performed each 24 weeks were review of symptoms and Adverse Events (AE), examination, weight, VL, full blood count, routine serum chemistries and urinalysis. Additional assessments performed every 48 weeks were diet and exercise history, questionnaire completion, CD4 count, fasting glucose and lipids, HbA1c, ELF score, Fibroscan. An optional CT scan of liver and spleen was performed at 96 weeks. Self-reported adherence noted on diary cards and pill counts by the pharmacist of returned MVC doses were recorded every visit. AE severity and laboratory abnormalities was assessed using the National Institute of Allergy and Infectious Diseases Division of AIDS toxicity grading scale [31]. Laboratory analyses and imaging were performed at site.
The first participant was enrolled on 24/07/18 and the final participant completed the last study assessment on 08/11/21.

Outcome measures
The primary objective was to assess the safety, feasibility and acceptability of adding MVC to OBT in HIV-NAFLD. This was assessed through the following outcome measures and minimum target values to indicate feasibility: (1)

Analyses
Sample size. This was a pilot study evaluating feasibility, with the intention that results would be used to estimate the variability of the treatment effect of MVC on the ELF score. This in turn would inform the sample size calculation for an RCT to evaluate drug efficacy. We based our sample size justification for this pilot study on change in ELF score. In a previous longitudinal study in people with chronic liver disease, a unit increase of 1 in the ELF score was associated with a 2.5-fold increased risk of a liver-related event, adjusted for age and stage of fibrosis. A unit increase of 1 was therefore considered a clinically important entity [33]. Assuming the standard deviation (SD) of the ELF score is 1.12 [33], with 20 participants per group for the analysis, a difference in ELF of 1 point could be estimated with a 95% confidence interval (CI) from 0.3 to 1.7. Assuming an attrition rate of 33%, as reported in previous RCTs evaluating MVC [34], there was a target of 30 participants per group.
Statistical analyses. Reasons for screen failures are summarised in the CONSORT flowchart, according to the 2010 Statement extension to pilot and feasibility studies [35]. Baseline demographic and clinical characteristics are presented by treatment group and overall. Descriptive statistical analyses were performed. Continuous variables were described by their medians and interquartile ranges, and categorical variables by the frequency and percentage in each category.
Primary outcome measures are presented with 95% CI for the following measures, at 48 and 96 weeks: participant retention, the proportion of individuals for whom data are missing, the proportion of individuals with ARs and the level of self-reported adherence to MVC. The latter was calculated by (a) the proportion of study medication dispensed that was returned at the final study visit (b) the mean number of doses taken that were prescribed. The proportion of eligible individuals approached who were successfully recruited with 95% CI, and the monthly recruitment rate, are also presented.
For each secondary outcome, we estimated the difference in means between groups from baseline to 48 and to 96 weeks and the 95% CI around these differences [36]. A t-method, using 10,000 bootstrapped samples due to non-normality of data, was used for continuous variables and exact 95% binomial CIs for categorical variables [37]. Analyses followed intentionto-treat principles, using available data; no imputation of missing data was conducted. All analyses were performed using Stata version 17.0 [38].

Ethics
This study was approved by the London Dulwich Research Ethics Committee (Reference 17/ LO/2093). The authors did not have access to information that could identify individual participants during or after data collection. Study data will be made available on reasonable request to the Brighton & Sussex Clinical Trials Unit. The trial was registered on the ISRCTN Registry, registration number 31461655

Participant flow
Eighty individuals were referred for screening, 21 (26%) and 6 (8%) of whom were excluded prior to and following attendance at the formal eligibility assessment, respectively. The remaining 53 (66%) individuals entered the randomized treatment portion of the trial and were included in analyses; 23 (43%) and 30 (57%) were allocated to MVC+OBT and OBT, respectively. The flowchart and reasons for participant exclusion are shown in Fig 1. Five participants in the MVC+OBT group (5/23, 22%) and four in the OBT group (4/30, 13%) discontinued the trial before completing the final visit.

Baseline characteristics
Baseline demographic and clinical characteristics were broadly similar across groups (Table 1)

Primary outcome measures-assessment of progression criteria against predefined targets
All individuals considered eligible after screening consented to participate, exceeding the target of 50%. The average recruitment rate was 2.9 per month over 18 months, exceeding the target of 2 per month. Participant retention was 45/53 (85%, 95% CI (72%, 93%)) and 44/53 (83%, 95%CI (70%, 92%)) by weeks 48 and 96, respectively, exceeding the target of 65%. At weeks 48 / 96, data completeness was 94% / 96% respectively, exceeding the target of 80%. There were no Serious Adverse Reactions, with 5/23 (22%, 95% CI (5%, 49%)) participants reporting AR by week 48 and no new AR by week 96 (Table 2). This did not meet the target of less than 10%. Self-reported adherence could not be measured as diary cards were not consistently completed or brought to appointments. Therefore, adherence was identified from counting of returned pills and was 92% (SD 7%), exceeding the target of 90%.

Adverse events
Two SAEs were observed in the MVC+OBT group (community acquired pneumonia and gastro-oesophageal reflux disease with vomiting) and four in the OBT group (fatal COVID-19 pneumonia, urinary retention from benign prostatic hyperplasia, suicidal ideation, listeria  Table S3 in S1 File). Of five ARs, four were neurological: worsening of restless legs (n = 1), drowsiness with loss of appetite (n = 1) and dizziness (n = 2). The fifth individual experienced rash and vomiting possibly MVC-related. Two individuals with AR discontinued maraviroc, at 21 weeks and 81 weeks. All five ARs were of mild or moderate intensity and resolved. One additional individual discontinued maraviroc at week 14 due to disruption to their regimen from twice daily dosing. Table 3 shows the differences between treatment groups, comparing change from baseline to weeks 48 and 96 in clinical characteristics, including ALT, AST, lipids, HbA1c, CD4 count, ELF, LSM and CAP scores. The 95% CI were generally wide, indicating relatively imprecise estimation of the between-group difference in changes from baseline, due to the small sample size. In all cases, 95% CI included zero, consistent with there being no detectable differences between treatment groups. No cases of virological failure were observed. Blips, defined as a single VL 50-1000 c/ml followed by a VL<50c/ml, were seen in 5/23 (22%) and 3/30 (10%) individuals in the MVC+OBT versus OBT group respectively. Excluding blips identified at the baseline visit, the rate was 3/23 (13%) and 2/30 (7%), respectively.

Secondary outcome measures
Supplementary Table S4 in S1 File shows clinical characteristics comparing baseline, week 48 and 96 visits. Trends over time for most characteristics were similar comparing groups although two differences were observed. From baseline to week 96, decreases were observed in the median ALT (-8 IU/L) and LSM scores (-0.95kPa) for the MVC+OBT group versus increases in the OBT group (+4 IU/L and +0.65 kPa respectively). Consistent with this, median CAP score improvements over this period were greater in the MVC+OBT group (-59 dB/m versus -20 dB/ m). However, 95% CI values for all characteristics were consistent with there being no change.
For QoL outcomes, comparing change from baseline through weeks 48 and 96 between treatment groups, 95% CI were wide indicating imprecise estimation of between-group differences. In all cases, 95% CI included zero, consistent with there being no detectable betweengroup differences ( Table 4).

Protocol deviations
There were 263 protocol deviations due to missing a study assessment (96, 37%); COVID-19 pandemic related factors resulting in assessments out of window (67, 25%); assessment out of

Discussion
These data provide preliminary evidence that add-on maraviroc as therapy for NAFLD without cirrhosis in PWH on effective cART may be safe, acceptable and feasible. We recruited 90% of our target of 60 individuals, and, of those referred for possible participation, 66% were randomised, including all who met eligibility criteria at screen. Participant retention until week 96 was acceptable, achieving~80%, despite the unprecedented circumstances of the SARS-CoV-2 pandemic. Adherence to maraviroc, taken twice daily by almost all participants, was high (>90%). The adverse reaction rate of 22% was numerically greater than the 10% preselected target; however, this figure fell within the 95% CI around 22% of 5%-49% indicating Table 3. Difference in change from baseline in metabolic and liver parameters for the maraviroc + OBT versus OBT group with bootstrapped 95% confidence intervals. the uncertainty as to whether or not there was an increase in AR compared to the target. Taken in conjunction with findings that all adverse reactions resolved, including in the 9% who discontinued maraviroc, and that there were no serious adverse reactions, no clear evidence of a significant safety concern was identified It was notable that blip rates were numerically greater in the MVC+OBT versus OBT arms (22% versus 10%), but if baseline visit blips were excluded, these became 13% and 7% respectively. Although this remained higher in the MVC+OBT group, both rates are comparable to that seen in the clinic population for one of the participating sites with available published data (~10%) [39]. We observed differences in the changes of two participant characteristics during follow up between the two treatment groups. ALT and LSM scores improved in the MVC+OBT but worsened in the OBT group over 96 weeks. CAP scores declined in both groups but by a numerically greater level in the MVC+OBT group. These findings raise the possibility of an improvement in liver fat and fibrosis. However, the 95% CI for the differences contained zero so were consistent with there being no detectable differences, and the trial was not powered to evaluate for differences in these characteristics. A future RCT of add-on maraviroc with a larger sample size should assess for any differences in these outcomes. There was a high level (>80%) of completion of questionnaires to assess QoL outcomes for CLDQ-NAFLD and SF36, but <50% completion for WPAI:SHIP. This was related to lack of responses to work productivity questions only, likely due to a combination of some participants being unemployed plus missing data. CLDQ-NAFLD and SF36 would therefore be more suitable for inclusion in a larger trial. Regarding QoL outcomes, 95% CI for differences between groups contained zero which was consistent with there being no detectable difference. Few individuals undertook optional CTs, either as this was not offered by sites or participant choice. This may be because CT is less acceptable than ultrasound for NAFLD follow up due to the increased radiation exposure and should not be included in a future RCT. The alternative imaging modality of Magnetic resonance imaging (MRI)-estimated proton density fat fraction (PDFF) is expensive and not routinely available.

Week Number of participants Difference between MVC+OBT / OBT groups in change from baseline 95% LCL 95% UCL
Limited data on the potential efficacy of maraviroc as a treatment for NAFLD are available from previous studies. In a retrospective cohort in PWH, 74 individuals receiving maraviroc were compared to 312 who had never been exposed to maraviroc, matched for age, sex and CD4 count nadir. In the non-maraviroc group, a significant association was found between a marker of inflammation (hsCRP) and lipoprotein levels (LDL, TG, TC) at baseline and after 3 years. By contrast, in the maraviroc group, this relationship was observed at baseline but lost after 3 years. The incidence of non-AIDS-defining disease was lower in the maraviroc group but this was not statistically significant. The authors speculated that CCR5 inhibition may offer protection against a lipid-dependent inflammatory process [17].
In a phase III trial, PWH -NAFLD were randomised into four groups: OBT (n = 24), ± maraviroc (n = 23), metformin (n = 21) or maraviroc with metformin (n = 22). Hepatic fat scores were measured at baseline and week 48 using MRI-PDFF. Maraviroc ± metformin was found to be safe with acceptable tolerability but did not reduce liver fat scores compared to no adjunctive treatment, and LSM scores were not presented [40]. In a proof of concept, single arm, prospective study of OBT plus add-on maraviroc with HIV-NAFLD, liver biopsies were compared at baseline and week 48. No significant impact on liver histology was observed including an absence of change in CD4+, CD8+ or CD68+ immune cell infiltrates, NAFLD Activity Score, ballooning and steatosis or lipid metabolism, although only thirteen individuals were included [41].
We instituted a number of measures in response to the COVID-19 pandemic. First, time windows for visits were lengthened from ±1 to ±8 weeks to provide flexibility for staff redeployed to COVID-19 responsive work and for participants experiencing difficulty in accessing clinical sites. Second, virtual visits were permitted for follow up visits, excluding week 48 and 96. This was to provide focus on high completeness of the final dataset whilst enabling participants to avoid travel and clinical contact while self-isolating. Third, we regularly sought feedback from sites to identify problems early and work towards finding local solutions for keeping participants engaged, for example, mailing out IMP.

Limitations
We used non-invasive assessment methods for hepatic steatosis and fibrosis, whilst the gold standard, liver biopsy, would have permitted more accurate classification of disease. However, recruiting a sufficient sample of PWH and mild NAFLD to a randomised study requiring consecutive liver biopsies presents feasibility challenges. We did not control for all ART classes, and emerging data have highlighted the possible association of INSTI and TAF with NAFLD progression, as well as a possible protective role for tenofovir disoproxil, although data are conflicting [42][43][44]. It would have been challenging to stratify for all ART agents possibly associated with risk of or protection from NAFLD in this small pilot study, including PIs, INSTI, TAF and TDF. Further, much of the emerging data highlighting an association between INSTI and/or TAF with NAFLD, and the negative association between NAFLD and TDF, became available after the last participant to this study had been recruited. A further limitation was that duration of HIV regimen was not controlled prior to participation. Additionally, 5/30 (17%) and 1/23 (4%) individuals in the OBT and MVC+OBT groups, respectively, switched one of more components of their backbone whilst on study; five were in-class switches and one an NNRTI to INSTI switch.Our cohort was~90% white and male and therefore applicability to other groups, particularly women and black and other minority ethnic groups, is unclear.

Considerations for an efficacy RCT of add-on MVC in HIV-NAFLD
Given conflicting data for associations between NAFLD development and different ART classes [42][43][44], eligibility criteria for a future RCT assessing efficacy of add-on MVC in HIV--NAFLD would need to carefully consider OBT regimen, for example, stratifying by PI, INSTI and TAF-containing therapy. Additional stratification may be required according to co-medications with proven or possible activity against NAFLD, such as tesamorelin, vitamin E or statins [9,10,45]. Given the uncertainty as to whether or not there was a greater AR rate compared to the pre-selected threshold, careful monitoring of ARs would also be needed. Although the blip rate whilst receiving MVC+OBT was comparable to that in the clinic population, the greater blip rate in the MVC+OBT versus OBT group highlights that close viral load monitoring may also be advised. Finally, given the need in this pilot for 7 sites to achieve 90% of the target, and considerations in a future trial around stratification by ARV backbone, to achieve a sample size of 103 participants per group in an efficacy RCT with 90% power, assuming a Cohen's D of 0.5 (i.e. a medium effect size) and attrition rate of 17%, a multi-national trial would have greatest likelihood of recruiting to target.

Conclusions
This study provides preliminary evidence that add-on maraviroc as therapy for HIV-NAFLD may be safe, feasible and acceptable. Although there were differences in the trend for ALT and LSM scores, with improvement in the MVC+OBT versus OBT group, the 95% CI contained zero, which was consistent with there being no detectable differences, and the study was not powered to evaluate changes in these characteristics. Overall, these data support a larger RCT assessing efficacy of add-on maraviroc on hepatic steatosis and fibrosis in PWH and NAFLD.